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Transient de-protonation of the chromophore affects protein dynamics proximal and distal to the linear tetrapyrrole chromophore in phytochrome Cph1.

M. Sadeghi,Jens Balke,Constantin Schneider,S. Nagano,Johannes Stellmacher,G. Lochnit,C. Lang,C. Weise,J. Hughes,U. Alexiev

Published 2020 in Biochemistry

ABSTRACT

Phytochromes are biological red/far-red light sensors found in many organisms. Prototypical phytochromes including Cph1 from the cyanobacterium Synechocystis 6803 act as photochemical switches that interconvert between stable red (Pr) and metastable far-red (Pfr) - absorbing states induced by photoisomerization of the bilin chromophore. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY) and the C-terminal transmitter (output) module, usually a histidine kinase, as in the case of Cph1. The chromophore deprotonates transiently during the Pr → Pfr photoconversion in association with extensive global structural changes required for signal transmission. Here, we performed equilibrium studies in the Pr-state, involving pH-titration of the linear tetrapyrrole chromophore in different Cph1 constructs, and measurement of pH-dependent structural changes at various positions in the protein using picosecond time-resolved fluorescence anisotropy. The fluorescent reporter group was attached at positions 371 (PHY domain), 305 (GAF domain) and 120 (PAS domain), as well as at sites in the PAS-GAF bidomain. We show direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains. Our results suggest that chromophore deprotonation is closely associated with a higher protein mobility (conformational space) both in proximal and distal protein sites, implying a causal relationship that might be important for the global large protein arrangements and thus intramolecular signal transduction.

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